The urokinase plasminogen activator (uPA) is a serine protease that interacts with its cell surface receptor (uPAR) providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. The uPA:uPAR complex converts plasminogen into plasmin which is known to degrade various matrix glycoproteins as described in Ellis et al, J. Biol. Chem. 264: 2185–2188 (1989), Vassili et al, J. Clin. Invest. 88: 1067–1072 (1991), and Mignatti and Rifkin, Physiol. Rev. 73: 161–195 (1993). The simaltaneous expression of uPA and its receptor has been associated with localized plasminogen activation and pericellular matrix degradation during directed cell migration of normal and tumor cells.
The urokinase receptor (uPAR) is a 283 amino acid glycosylphosphatidyl-inositol (GPI)—anchored receptor protein of urokinase and vitronectin which appears to be a triplication of a 90 amino acid domain as described in Plough, and Ellis, FEBS Lett. 349:163–168 (1994) and Roldan et al, EMBO J. 9: 467–474 (1990). Proteolysis of uPAR can yield fragments composed of domain 1 and domains 2–3, and subsequent analysis has shown that disulfide bonding pattern of domain 1 is completely internal to the domain, as described in Plough et al, J. Biol. Chem. 268:17539–17546 (1993), and Kieffer et al, Biochem. 33:4471–4482 (1994).
The migration and invasion of cells appear to require cell surface localized proteolysis and adhesion to specific components of the extracellular matrix. These processes are necessary for many normal and pathological processes, including tissue remodeling, embryo implantation, angiogenesis, and tumor cell invasion and metastasis as described in Fazioli et al, Trends Pharimacol. Sci. 15:25–29(1994), and Mignatti et al, Physiol. Rev. 73:161–195 (1993). Important components of the cell surface proteolytic and cellular adhesion cascades are the plasminogen activator/plasmin system, matrix metalloproteinases, and integrins, as described in Felding-Habermann et al, Curr. Biol. 5 864–868 (1993). Adhesion to the extracellular matrix component vitronectin has been reported to correlate with UPAR expression, and uPA binding sites and vitronectin receptors have been shown to colocalize on HT1080 cells, as described in Waltz et al, J. Biol. Chem. 269: 14746–14750 (1994), and Ciambrone et al, J. Biol. Chem. 267: 13617–13622 (1992). More recently it has been demonstrated that uPAR can function as a cell adhesion receptor for vitronectin in a uPA dependent manner as described in Wei et al, J. Biol. Chem. 269: 32380–32388 (1994).
Early experiments using chemical cross-linking suggested that the first domain of uPAR was sufficient for high affinity binding of uPA, however, subsequent work has shown that an intact 3-domain molecule is required, and that additional binding determinants in domains 2 and 3 are likely involved, as described in Plough et al, Biochem. 3: 8991–8997 (1994). The undefined interactions may be with the uPA EGF-like domain or indirect interactions affecting the conformation of domain 1. Previous work has been unsuccessful in distinguishing whether domain 2 and 3 has measurable affinity for uPA, because of the difficulty of separating domain 2 and 3 from trace amounts of full length uPAR as described in Plough et al, Biochem. 3: 8991–8997 (1994).
The uPA:uPAR system has been identified as promoting pericellular proteolysis, and functions attributable to uPAR include cell migration, adhesion and mitogenesis. It would be desirable, therefore, to elucidate the function of domains 2 and 3 of uPAR.